Summary/Abstract JAK2 is a tyrosine kinase that is activated by two-thirds of the cytokine/hematopoietin receptor superfamily. Therefore, the contribution of JAK2 to different pathologies is increasingly appreciated. There is a gap in our understanding of a comprehensive mechanism of JAK2 regulation in normal and pathological conditions. The long-term goal of our research is to establish important insight into the fundamental role of centrosomal localization of JAK2 in the regulation of cell proliferation. The overall objective of the current application is to characterize a JAK2-containing complex localizing to the centrosome and regulating cell proliferation. The central hypothesis is that JAK2 is a novel member of a centrosome associated complex and that this localization regulates cell cycle and cell proliferation. The rational for the proposed research is that activated JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. Guided by strong preliminary data, the central hypothesis will be tested by pursuing the following specific aims: Specific Aim 1 will test our working hypothesis that, in response to prolactin (PRL) and interferon ? (IFN?), activated JAK2 translocates to the centrosome to regulate cell proliferation. Under the first aim, we will identify which part of JAK2 targets it to the centrosome (centrosomal localization sequence, CLS) and the impact of the CLS on JAK2-dependent signaling in response to PRL and IFN?. Specific Aim 2 will determine whether JAK2 complexes with the serine/threonine kinase PAK1 on centrosomes to regulate mitosis and/or G2-M transition and elucidate the underlying mechanism(s). Under this aim, we will determine whether centrosome-localized JAK2 phosphorylates Tyr(s) 153, 201 and 285 of PAK1 which then facilitates activation of Polo-like kinase 1 (Plk1) and Aurora A on the centrosome to regulate G2-M progression and cell proliferation. This proposal is conceptually innovative because it is expected to vertically advance and expand our understanding of how JAK2/PAK1 complex regulates cell proliferation and the unique role of centrosome in this process. The proposed research is significant because the elucidation of JAK2- mediated intracellular signaling pathways will result in better understanding of the mechanisms involved in regulation of cell proliferation and provide insight into future therapeutic approaches to human cancer.